PROJECT SUMMARY Melioidosis is a tropical infection caused by inhalation, inoculation, or ingestion of the Gram-negative facultative intracellular pathogen and Tier 1 select agent Burkholderia pseudomallei (Bps). The clinical manifestations of melioidosis are protean but pneumonia is present in over 50% of cases and more than doubles the risk of death. As a result, the airborne route of infection with Bps is of greatest concern in a bioterrorism scenario. Melioidosis is often severe and treatment is hampered by difficulty eradicating the pathogen due to its intrinsic antimicrobial resistance. Worldwide, 165,000 cases of melioidosis are estimated to occur each year; 85,000 (52%) of these patients die. In northeast Thailand where the disease is endemic and well recognized by clinicians, 40% of patients die despite appropriate antibiotic therapy. Development of novel, targeted, and effective therapeutics to reduce this burden of disease is therefore of paramount importance. Achieving this objective necessitates a better understanding of the host-pathogen interaction during melioidosis, especially the host-pathogen interaction in the lung. Bps infects a variety of phagocytic and non- phagocytic mammalian cells and thrives in the intracellular environment. In these cells Bps successfully evades or suppresses the immune response. Prior studies, mostly using murine cells and murine models, indicate that resistance to Bps infection is dependent on activation of the host inflammasome. However, preliminary data generated in primary human cells indicate that human alveolar macrophages do not depend on the inflammasome for initial control of Bps. In contrast, in human bronchial epithelial cells, Bps overcomes host defenses to proliferate with impunity. The hypothesis of this project is that whereas Bps may overcome the inflammasome in bronchial epithelial cells, in alveolar macrophages successful host defense occurs independent of the inflammasome. The goal of this project is to comprehensively evaluate the distinct role of the inflammasome in these primary human lung cells. This host defense mechanism will be evaluated in alveolar macrophages (Aim 1), in bronchial epithelial cells (Aim 2), and in human lung tissue ex vivo (Aim 3). This investigation is a not only an essential step towards improving our understanding of the striking virulence of this pathogen in the lung, but establishes a robust and flexible isolated human lung platform for further research focused on the interaction of the human host with Bps.